Design, synthesis and evaluation of the antibacterial activity of new Linezolid dipeptide-type analogues

Article abstract of DOI:10.1016/j.bioorg.2019.103483

Worldwide studies towards development of new drugs with a lower rate in emergence of bacterial resistance have been conducted. The molecular docking analysis gives a possibility to predict the activity of new compounds before to perform their synthesis. In this work, the molecular docking analysis of 64 Linezolid dipeptide-type analogues was performed to predict their activity. The most negative scores correspond to six Fmoc-protected analogues (9as, 9bs, 9bu, 10as, 10ax and 10ay) where Fmoc group interacts in PTC for Linezolid. Twenty-six different Fmoc-protected Linezolid dipeptide-type analogues 9(as-bz) and 10(as-bz) were synthesized and tested in antimicrobial experiments. Compounds 9as, 9ay, 9ax, 10as, 10ay and 9bu show significant activity against group A Streptococcus clinical isolated. Analogue 10ay also display high activity against ATCC 25923 Staphylococcus aureus strain and MRSA-3, MRSA-4 and MRSA-5 clinical isolates, with MIC values lower than Linezolid. The highest activity against multidrug-resistant clinical isolates of Mycobacterium tuberculosis was exhibited by 9bu. Finally, a cytotoxicity assay with ARPE-19 human cells revealed a non-cytotoxic effect of 9bu and 10ay at 50 and 25 μM, respectively.

Full text of DOI:10.1016/j.bioorg.2019.103483

Journal Pre-proofs
Design, synthesis and evaluation of the antibacterial activity of new Linezolid
dipeptide-type analogues
G.D. García-Olaiz, Eleazar Alcántar-Zavala, Adrián Ochoa-Terán, Alberto
Cabrera, Raquel Muñiz-Salazar, Julio Montes-Ávila, Alex J. Salazar-Medina,
Efrain Alday-Noriega, Carlos Velazquez, José L. Medina-Franco, Rafael
Laniado-Laborín
PII:
S0045-2068(19)31375-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103483
YBIOO 103483
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 September 2019
22 November 2019
25 November 2019
Please cite this article as: G.D. García-Olaiz, E. Alcántar-Zavala, A. Ochoa-Terán, A. Cabrera, R. Muñiz-Salazar,
J. Montes-Ávila, A.J. Salazar-Medina, E. Alday-Noriega, C. Velazquez, J.L. Medina-Franco, R. Laniado-
Laborín, Design, synthesis and evaluation of the antibacterial activity of new Linezolid dipeptide-type analogues,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103483
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Design, synthesis and evaluation of the antibacterial activity of new
Linezolid dipeptide-type analogues
G. D. García-Olaiz,1† Eleazar Alcántar-Zavala,1† Adrián Ochoa-Terán,1* Alberto Cabrera,1
Raquel Muñiz-Salazar,2 Julio Montes-Ávila,3 Alex J. Salazar-Medina,4 Efrain Alday-
Noriega,5 Carlos Velazquez,5 José L. Medina-Franco,6 Rafael Laniado-Laborín,7,8
1
Centro de Graduados e Investigación en Química, Tecnológico Nacional de
México/Instituto Tecnológico de Tijuana. Tijuana, B. C. México.
2
Laboratorio de Epidemiología y Ecología Molecular, Escuela Ciencias de la Salud,
Universidad Autónoma de Baja California, Unidad Valle Dorado. Ensenada, B. C. México.
3
Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa. Culiacán,
Sin. México.
4 Cátedras Conacyt - Departamento de Investigación en Polímeros y Materiales, Universidad
de Sonora. Hermosillo, Sonora, México.
5 Department of Chemistry-Biology, University of Sonora. Hermosillo, Sonora, México.
6
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de
México. Ciudad de México, México.
7 Clínica y Laboratorio de Tuberculosis, Hospital General de Tijuana, ISESALUD. Tijuana,
B. C. México.
8 Facultad de Medicina y Psicología, Universidad Autónoma de Baja California. Tijuana, B.
C. México.
† These authors equally contribute to this work
* Correspondence e-mail: ochoa@tectijuana.mx
1

Abstract
Worldwide studies towards development of new drugs with a lower rate in emergence
of bacterial resistance have been conducted. The molecular docking analysis gives a
possibility to predict the activity of new compounds before to perform their synthesis. In this
work, the molecular docking analysis of 64 Linezolid dipeptide-type analogues was
performed to predict their activity. The most negative scores correspond to six Fmoc-
protected analogues (9as, 9bs, 9bu, 10as, 10ax and 10ay) where Fmoc group interacts in
PTC for Linezolid. Twenty-six different Fmoc-protected Linezolid dipeptide-type analogues
9(as-bz) and 10(as-bz) were synthesized and tested in antimicrobial experiments.
Compounds 9as, 9ay, 9ax, 10as, 10ay and 9bu show significant activity against group A
Streptococcus clinical isolated. The analogue 10ay also display high activity against ATCC
25923 Staphylococcus aureus strain and MRSA-3, MRSA-4 and MRSA-5 clinical isolates,
with MIC values lower than Linezolid. The highest activity against multidrug-resistant
clinical isolates of Mycobacterium tuberculosis was exhibited by 9bu. Finally, a cytotoxicity
assay with ARPE-19 human cells revealed a non-cytotoxic effect of 9bu and 10ay at 50 and
25 mM, respectively.
Keywords: Linezolid analogues, dipeptide-type, docking, antibacterial activity, organic
synthesis
1. - Introduction
The research of new antibiotics is essential due to the resistance developed by bacterial
strains towards conventional antibiotics. The most important bacterial pathogens for humans
evolved resistance and multi-resistance as result of a wide spread use of antibiotics through
the last 70 years.1 In United States, multi-resistant bacteria affect two million people per year,
resulting in at least 23,000 dies per year.2 Recently, World Health Organization (WHO)
release a list of 12 antibiotic-resistant priority pathogens, such as Staphylococcus aureus,
Streptococcus pneumoniae and Mycobacterium tuberculosis.3 Oxazolidinones are a new
group of antibiotics approved in 35 years.4 Linezolid, an oxazolidinone based drug, is one of
these outstanding therapeutic compounds with a different action mechanism (Figure 1).1,5
Linezolid inhibits protein synthesis of bacterias in the initiation phase. An X-ray crystal
2

structure analysis showed its insertion in the 50S ribosome subunit through a hydrogen bond
between the acetamidomethyl NH and phosphate of G2540 in the Haloarcula marismortui
strain. In addition, an interaction between the C2487 and the fluorophenyl group is observed.6
Linezolid
a
d
b
c
Figure 1. Chemical structure of Linezolid and different analogues modified in the C5 (a), A
ring (b), B ring (c) and C ring (d).
Linezolid is active against most of Gram-positive bacteria and vancomycin-resistant
enterococcus. However, there are current reports of resistance to Linezolid in Staphylococcus
epidermidis, Staphylococcus haemolyticus and Staphylococcus aureus strains, especially in
prolonged stay patients with nosocomial infections. Gram-positive bacteria usually develop
resistance to Linezolid as result of mutation in G2576T, the Guanine base is replaced by
Thymine in the 23S ribosome subunit.7,8
Since the emergence of resistant strains to inhibitory activity of Linezolid, structural
analogues have been synthesized to improve its effectiveness.9-11 The original structure has
been modified in four sites: oxazolidinone (ring A)12, fluorphenyl group (ring B)13,
morpholine (ring C)14 and C5 substituent15 (Figure 1). Structural modifications demonstrate
that the presence of an N-aryl group is necessary to the biological activity, as well as, the S
configuration at C5 in the oxazolidinone ring, which is essential to induce a positive influence
in the antibacterial activity. Morpholine group provides a safe pharmacokinetic profile due
to the presence of an electron donating group in the aromatic ring. Finally, the presence of a
fluorine atom attached to an aromatic ring have shown to improve the antibacterial activity
of Linezolid analogues.16-22
3

Nowadays, the design and development of new drugs are based on the benefits of low
molecular weight drugs (less than 500 Da), which have conformational restriction, good
membrane permeability, metabolic stability and oral bioavailability, combined with the
selectivity in the action site and high potency of biological compounds (higher than 5000
Da).23 The inclusion of peptide moieties in the structure of conventional drugs is an optimum
strategy to improve their biological activity. These new compounds recognize as “peptide
drugs” (structures with less than 50 amino acids) proved to be more active than their drug
precursors.24
Pan et. al. synthesized dipeptide derivatives of dehydroabietic acid,24 wherein the
carboxylic group was modified with a dipeptide moiety showing a higher level of inhibitory
activity against cancer cell lines, and a more potent effect than commercial anticancer drug
5-fluorouracil. Moreover, the structural inclusion of dipeptide moieties in conventional drugs
has been develop in the antimicrobial area. These new antibiotics reduce bacterial resistance
due to the rapid inhibition of bacterial growth by physical disruption of cell membranes.24
The functional groups in a dipeptide moiety establish more interactions, resulting in an
improved interaction in the action site.25 Therefore, antibacterial activity of Linezolid may be
enhanced including a dipeptide-type moiety to increase the number of interactions in the
PTC. In fact, the enhanced antimicrobial activity of Tedizolid, a new Linezolid analogue
drug, is attributed to its interaction in the action site through four-hydrogen bonds.26
Some Linezolid analogues have been synthesize modifying the C5 substituent with
different amphiphilic segments such as amino acids (Lysine or Arginine), hydrophobic
chains and cationic segments.27 These analogues display activity against Gram-negative
strains as E. coli and S. enterica with MIC values of 8 μg/mL (Linezolid was 64 μg/mL).
However, these compounds were inactive in a serum-blood (1:1) solution. This result is due
to presence of negative charges in proteins and other macromolecules present in serum and
blood, which bind to cationic molecules, and avoid their transport into bacterial membrane.
Some thiazolidinone dipeptide type analogues have been synthesized and tested as inhibitors
of type III secretion system in Salmonella enterica serovar Typhimurium. The IC50 obtained
for the most analogues modified as dipeptides, were lower than the obtained with the
thiazolidinone precursor. 28
4

Previously, we reported the diasteroselective synthesis of ten dibenzylamino
oxazolidinones by chemical modification of five different a-aminoacids.29 The in vitro
antibacterial assays were performed with 12-methicillin resistant Staphylococcus aureus
clinical isolates. The oxazolidinone derived of L-alanine exhibited antibacterial activity in
the Kirby-Bauer diffusion susceptibility protocol at 6.6 μg and a MIC value of 12.5 mg/mL
in the microdilution method.30
Here we report the molecular docking and antibacterial activity of Linezolid
dipeptide-type analogues, which preserve A, B and C rings of Linezolid, with a variation in
A ring configuration (R or S) (Figure 2). The construction of A ring is achieve by chemical
modification of L-Alanine (R1= methyl) and L-Phenylalanine (R1= benzyl), leading to
oxazolidinone diasteromers (S,R and S,S).31 The combinatorial coupling with a Fmoc-
protected amino acid (AA) leads to S,R,S and S,S,S diasteromers. L-Alanine and L-
Phenylalanine were chose to build the A ring considering the antibacterial activity of the
oxazolidinone derivative,30 and to evaluate the effect an aromatic ring in the molecular
coupling and antibacterial activity. The amino acids selected as AA have different structural
characteristics such as aromatic and heteroaromatic rings (Phe, Trp, His, Tyr), aliphatic (Leu)
and heteroaliphatic (Thr, Cys, Ser) groups, further, the protective group is Fmoc. The
combination of the four-diasteromeric oxazolidinones with the AA2 amino acids leads to 32
compounds. The total deprotection of these compounds finally reached a database of 64
Linezolid dipeptide-type analogues for testing in the molecular docking analysis.
5

Linezolid base structure
Linezolid dipeptide-type analogues
AA
Figure 2. Combinatorial strategy to design the Linezolid dipeptide-type analogues.
The hypothesis of this study lays in structural modifications of Linezolid with a-
amino acids to maintain or increase of their activity, as well as, retard the bacterial resistance.
These Linezolid dipeptide-type analogues have the amide group required to establish a
typical hydrogen bond interaction, and different R1 and R2 groups, which may display
different interactions in the action site.
3. – Results and discussion
3.1 Molecular docking studies
The molecular docking of Linezolid and analogues was perform into the
crystallographic structure of ribosomal RNA (rRNA) of E. coli. The crystallographic
structure was reported at 3.5 Å of resolution for Schuwirth et al.32 The Phosphoryl
Transferase Center (PTC) in 4V4Q is located in the E. coli 23S rRNA subunit. The binding
pocket is made up of a group of nucleotides within of 4.5 Å in relation with the atoms of the
ligand. This structure was selected to perform the molecular docking deleting ligands such
as magnesium ion, water molecules, ribosomal proteins and a repetitive chain of rRNA.
6

The geometry of molecules was optimized with the QuickPrep application
implemented in MOE program,33 which adds hydrogen and establishes protonation states,
fixes distant atoms of receptor from the molecule, and leaves free nearby atoms. First, the
molecular docking of Linezolid was performed considering flexible the atoms of molecule
and receptor. An AMBER force field (AMBER10:EHT) was selected to produce an
homogeneous distribution of the low energy conformations (500 interactions in total with 30
consecutive attempts to find a better solution) and the best coupling result was selected.34
Molecular docking of Linezolid with MOE program shows a hydrogen bond
interaction with G2505. In the boundaries of PTC are G2505 and U2506 surrounding the
guest near to oxazolidinone ring, as was observed in docking with Linezolid by Shaw et al.26
More nucleotides that surround the guest are A2503, A2451 and C2452. However, an
opposite orientation of fluorine atom was found in our model (Figure S1). The coupling score
value is -7.21 kcal/mol and a redocking give a RMSD value of 1.17 Å. A second Linezolid
molecular docking was performed with LEDOCK 2018 program,35 in order to verify the
consistency and reproducibility of the pose reported by MOE 2018. As depicted in Figure
S2, this analysis predicted a similar pose than MOE and there is perfect overlap of both poses
obtained by the two programs. The score value obtained by LEDOCK program was -6.02
kcal/mol. The different orientation of fluorine atom in our model is presumably due to the
refinement in the programs algorisms looking for predictions that are more accurate and the
use of a different crystal structure of the rRNA for E. coli.
Once the molecular docking of Linezolid was validated with MOE and LEDOCK,
the molecular docking of 64-Linezolid dipeptide-type analogues was perform into the
crystallographic structure of rRNA in MOE 2018. The analysis showed 30 guests occupying
the PTC and showing a hydrogen bond interaction between amide group of Linezolid and the
G2505 nucleotide of the rRNA fragment. Noteworthy, the most negative scores (in kcal/mol)
correspond to the six protected analogues (9as, 9bs, 9bu, 10as, 10ax and 10ay) where the
Fmoc and the other protecting groups interacting in PTC (Table 1). The score value obtained
after several calculations is relate to the quality of the coupling, where the lower scores
indicate the most favorable couplings. Based on the docking results and a previous report of
antibacterial activity in E. coli and S. aureus strains and docking studies of N-protected
7

(Fmoc-protected) hydroxyamic acids,36 this research was focused in the study of the
Linezolid dipeptide-type analogues bearing non-polar protecting groups.
Table 1. Score of the molecular coupling experiment.
Compound Diasteromer R1
AA
Phe
Phe
Leu
Phe
Cys
Hys
Score (kcal/mol)
-10.42
R,S,S
S,S,S
R,S,S
R,S,S
R,S,S
S,S,S
CH3
CH3
Bn
9as
10as
-9.41
-9.38
9bu
Bn
-10.23
9bs
CH3
CH3
-9.85
9ax
-10.67
10ay
Linezolid
-7.21
Standard deviation 1.05
Average -8.4289
The docking of analogue 9as (green color) in PTC is an example of a perfect
superposition with Linezolid (yellow color). This guest establish a typical hydrogen bond of
amide NH with G2505, similar to Linezolid. In addition, there is a σ-π interaction between
the fluorene ring (Fmoc group) and hydrogen in C2 of the purine in A2062 (Figure 3).
a)
b)
Figure 3. a) Molecular docking of 9as (blue) and Linezolid (magenta) in PTC and b)
interactions map of 9as.
8

Some analogues as 9bu present a non-Linezolid like space arrangement, but they
display more main interactions in PTC (Figure 4). Figure 4 shows the molecular docking of
9bu, a π-π interaction between the benzyl group and A2451 nucleotide base, a σ-π interaction
between Fmoc group and the G2505 nucleotide base and two additional hydrogen bond
interactions with the same nucleotide are observed.
a)
b)
Figure 4. a) Molecular docking of 9bu (blue) and Linezolid (magenta) in PTC and b)
interactions map of 9bu.
Analogue 10ay presents a Linezolid shifted pose (yellow color) in the active site, and
the characteristic hydrogen bond between amide NH and G2505 is not observed. The
structure of 10ay (yellow color) shows different interactions with G2061 and G2505
nucleotides in the molecular docking analysis. Apparently, there is a hydrogen bond with the
Fmoc methylene group and a π-π interaction with the imidazole and G2505 nucleotide.
Additionally, a hydrogen bond interaction was observed between the carbonyl of the A ring
and G2061 nucleotide (Figure 5).
9

a)
b)
Figure 5. a) Molecular docking of 10ay (blue) and Linezolid (magenta) in PTC and b)
interactions map.
The molecular docking of the non-protected 10ay analogue shows a shifted
Linezolid-like pose (Figure 6). The hydrogen bond between the amide NH group of Linezolid
and the G2505 phosphodiester is not present either, but the interaction of the G2061
nucleotide and the A ring carbonyl acting as hydrogen acceptor remains present. Noteworthy,
the p-p interaction of the unprotected imidazole ring with the guanine in the G2505
nucleotide is no longer present and two new hydrogen bond interactions between the amide
carbonyl and free amine of the histidine with the G2505 nucleotide are established. The score
value of these unprotected anologue is -7.93 kcal/mol, which also predict a favorable activity.
10

a)
b)
Figure 6. a) Molecular docking of non-protected 10ay analogue (purple) and Linezolid
(yellow) in PTC and b) interactions map.
In summary, the docking analysis of the Linezolid-dipeptide type analogues predicts
additional interactions that favor their coupling in the PTC with score values
thermodynamically more favorable. The protecting groups influence significantly in the
spatial arrangement and in the nature and number of interactions in the PTC.
3.2 Chemistry
A library of 26 Linezolid dipeptide-type analogues was prepared following a three
step syntheses pathway as depicted in Scheme 1. First, the chemical modification of L-alanine
(a) and L-phenylalanine (b) leads to the diastereomeric oxazolidinone pair S,R-2 and S,S-3
from each amino acid, respectively (Scheme S1).29,30 A N-aryl coupling was achieved with
131,37 and 2(a-b) and 3(a-b), respectively, using potassium carbonate, N,N-
dimethylethylamine and copper iodide to obtain 4(a-b) and 5(a-b) in good yields. The
hydrogenolysis of these intermediates was carried out with H2, Pd/C in methanol to give 6(a-
b) and 7(a-b). Finally, amine intermediates were reacted with Fmoc-protected amino acids
8(s-z) (L-Phe, L-Leu, L-Tyr, L-Thr, L-Trp and L-Ser) using N,N-disopropylcarbodiimide
(DIC) and 1-Hydroxybenzotriazole (HOBt) in DMF.
11

Scheme 1. Reagents and conditions: (i) CuI, K2CO3, MeNH(CH2)NHMe, Toluene, 115°C,
72 h; (ii) H2, Pd/C 10%, MeOH, AcOH, 4 h; (iii) DIC, HOBt, DMF, 12 h.
1
The H NMR spectra of the oxazolidinones 2 and 3 show the signals for a A2B2C
aromatic system at 7.36, 7.26 and 7.16 ppm and two doublets at 3.70 and 3.44 ppm due to
presence of the dibenzyl amino protected group. There is a broad singlet at 6.45 ppm
corresponding to the oxazolidinone N-H group and at 3.55 and 3.22 ppm are present the two
signals for the diasterotopic methylene hydrogens. The signal of the stereocenter derived
13
from de amino acid is observed at 4.46 ppm. The C NMR spectra show a signal for the
carbonyl at 160.4 ppm, and at 79.9 and 44.8 ppm two signals assigned to the methine and
methylene carbons of the oxazolidinone, respectively. All these signals evidence the presence
of the oxazolidinone ring in their chemical structure. The signals corresponding the R1
substituent are also present. In addition, slight differences in the chemical shifts for some of
12

the signals in 2 and 3 confirm their different configuration (Figures S3-S10).29,30,38-41 The
FTIR spectra shows the vibration for the carbonyl in 1733 cm-1 (Figure S13).
The 1H NMR spectrum of 1 shows a multiplet signal at 7.21 ppm and a double of doublets at
6.82 ppm assigned to aromatic hydrogens. Signals at 3.87 and 3.07 ppm corresponds to
morpholine hydrogens. In the 13C NMR spectrum a doublet at 155.5 ppm with a JC-F = 249.3
Hz is assigned to the ipso fluorine carbon. A doublet at 113.9 ppm with J =8.7 Hz corresponds
to the ipso bromine carbon. Finally, two signals at 66.9 and 50.8 ppm correspond to
morpholine methylenes (Figures S11 and S12).
The 1H NMR spectra of 4 and 5 show signals corresponding to both precursors, with
exception of the N-H signal at 6.45 ppm (Figures S16-S23). The presence of three signals at
7.20, 7.00 and 6.91 ppm corresponding to fluorophenyl group, two multiplet signals at 3.86
and 3.05 ppm assigned to morpholine hydrogens and signals for two diasterotopic hydrogens
present in oxazolidinone at 3.82 and 3.57 ppm, confirm the presence of both fragments in
these molecules. In the 13C NMR spectra is present the oxazolidinone carbonyl signal at 154.2
ppm, and a doublet at 155.5 ppm with JC-F = 244.5 Hz for the ipso fluorine carbon. The ipso
amine carbon shifts to 136.3 ppm due to the bonding with nitrogen. These and the rest of
signals confirm the two fragments coupling. In the FT-IR spectrum, a carbonyl vibration is
present at 1748 cm-1 (Figure S14).
The signals corresponding to benzylic groups are no longer observed in NMR spectra of 6
and 7. A broad singlet at 4.31 ppm in 1H NMR spectra and a shift of the CH-N carbon from
56.8 to 48.3 ppm indicate the presence of a free amino group in these molecules (Figures S24
to S31).
Linezolid dipeptide-type analogues 9(as-bz) and 10(as-bz) display stretching vibration
bands at 1775 cm−1, 1760 cm−1 and 1684 cm−1 in their FTIR spectra, corresponding to Fmoc,
oxazolidinone and amide carbonyls (Figure S15). 1H NMR spectra show two broad singlets
at 6.30 and 5.45 ppm assigned to carbamate and amide hydrogens, respectively. A signal at
172 ppm corresponds to amide carbonyl and two signals at 154 ppm to carbamate and
oxazolidinone carbonyls. These spectroscopic data evidence the formation of dipeptide
analogues. Two-dimensional HSQC experiment were performed with these compounds, in
13

order to assign the rest of signals. NMR spectra (Figures S32 to S95) and total signal
assignation are provided in the supplementary material (Tables S1 to S12).
3.3 Antibacterial activity against Streptococcus Staphylococcus clinical strains.
The antibacterial efficacy of Linezolid dipeptide type analogues was determine by a
microdilution assay and expressed as the minimum inhibitory concentration (MIC).44-46 The
compounds 9as, 9ay, 9ax, 10as, 10ay, (R1=CH3) and 9bu (R1=Bn) showed significant
activities (MIC of 3.125 to 25 μg/mL) against group A Streptococcus clinical isolate (Figure
7).
10as
9as
10ay
9ay
9aw
9ax
9bu
Figure 7. Active Linezolid dipeptide-type analogues against Streptococcus and
Staphylococcus clinical strains.
There are more active analogues from L-alanine than L-phenylalanine (Table 2).
However, none of these show more potency than Linezolid (0.5 mg/mL) against group A
Streptococcus clinical strain. Interestingly, almost all active compounds have a R,S,S
configuration, but the most active compound 10ay is S,S,S, being twice more active than
9ay. This result indicates that stereochemistry influences the antibacterial activity of
Linezolid analogues.
14

Table 2. MICs of the Linezolid peptide-type analogues against drug-sensitive group A
Streptococcus clinical strain (MIC: µg/mL).
Compound Diasteromer
R1
AA
Phe
Thr
Cys
His
Leu
Phe
His
MIC
25.0
6.25
6.25
6.25
25.0
25.0
3.125
0.5
R,S,S
R,S,S
R,S,S
R,S,S
R,S,S
S,S,S
S,S,S
CH3
CH3
CH3
CH3
Bn
9as
9aw
9ax
9ay
9bu
CH3
CH3
10as
10ay
Linezolid
The analogue 10ay exhibits the highest activity against group A Streptococcus
clinical strain among other analogues, and it is active against ATCC 25923 Staphylococcus
aureus strain and MRSA-3, MRSA-4 and MRSA-5 clinical isolates, displaying lower MIC
values than Linezolid (Table 3). The effect of compound 10ay was determined by seeding
the well content in which the MIC value was determined in TSA medium and incubated for
20 h. The bacterial growth in culture media shows 10ay as bacteriostatic similar to Linezolid.
Table 3. Evaluation of the antibacterial activity of 10ay against ATCC and clinical strains
(MIC: µg/mL).
Compound
10ay
ATCC 25923 MRSA-3 MRSA-4 MRSA-5
1
3.125
0.5
3.125
4
3.125
4
3.125
8
6.25
8
Linezolid
Staphylococcus aureus ATCC 25923. Clinical isolates of Meticilin Resistant Staphylococcus aureus meticilin
resistant clinical strains (MRSA-3, MRSA-4, MRSA-5), group A Streptococcus (1).
Noteworthy, analogue 10ay shows the best score value in the molecular docking
analysis, as well as, performance in the antibacterial assay. The molecule of 10ay presents
three main interactions in the action site (Figure 4), while molecular docking of 9ay shows
two hydrogen bonding interactions with A2062 and G2505 (Figure S94). Both molecules
15

9ay and 10ay occupy the PTC of Linezolid (Figure 8a), but a different configuration in C5 of
A ring leads to a different spatial arrangement, as well as, number and type of supramolecular
interactions (Figure 8b). In 10ay the imidazole ring establishes a p-p interaction with G2505
and Fmoc group participates with two additional interactions. Analogue 9ay establishes a
hydrogen bond with imidazole ring, acting as acceptor and G2505 as donor, while the
interaction with Fmoc group is not present. The analogues 9ax and 9aw have significant
antibacterial activity consistent with their molecular docking analysis. The main interactions
of these guests into the Linezolid PTC are p-p and s-p interactions between Fmoc group and
G2505, as wells as, hydrogen bonding (Figures S97 and S98).
a)
b)
Figure 8. a) Molecular docking of Linezolid (magenta), 9ay (blue) and 10ay (yellow) and
in PTC. b) Overlap structures of 9ay (pink) and 10ay (green).
3.4 Antibacterial activity against multidrug-resistant Mycobacterium tuberculosis clinical
isolates.
First, all Linezolid dipeptide-type analogues were evaluated against three multidrug-
resistant Mycobacterium tuberculosis clinical isolates by a BACTEC MGIT 960 method in
a screening experiment at 125 mg/mL. The active analogues found in the experiment are L-
Phenylalanine derivatives 9bs, 9bw, 9bt, 9bu, 10bs and 10bw (Figure 9), and two L-Alanine
16

derivatives (9aw and 10aw). All analogues with Threonine as AA have shown significant
activity (9aw, 10aw, 9bw and 10 bw). The R,S,S and S,S,S analogues with a benzyl as R1 and
R2 (9bs and 10bs) also display activity. These results suggest that inhibitory activity may be
partially attribute to the presence of aromatic groups.
Table 4. Antibacterial activity evaluation of Linezolid analogues against multidrug-resistant
Mycobacterium tuberculosis clinical isolates (MIC: µg/mL).
Clinical isolate
Compound Diasteromer
R1
CH3
CH3
Bn
Bn
Bn
Bn
Bn
Bn
AA
Thr
Thr
Thr
Thr
Phe
Phe
Tyr
Leu
-
820 1143 1083
R,S,S
S,S,S
R,S,S
S,S,S
R,S,S
S,S,S
R,S,S
R,S,S
-
-
-
-
-
125
125
R
9aw
10aw
9bw
100 100
R
100 100
10bw
9bs
125 125
R
R
R
125
R
10bs
12.5
100
R
9bt
1.90 3.125
9bu
1.56 1.56 1.56
Linezolid
820: Resistant to isoniazid and rifampin
1143: Resistant to isoniazid, ethambutol and pyrazinamide
1083: Resistant to isoniazid, rifampin and streptomycin
A dilution experiment with the active analogues and Linezolid as control was
performed. The most active analogues are 9bt and 9bu with MIC values of 12.5 μg/mL and
1.90 μg/mL, respectively (Table 4). Analogue 9bu is the most active against two clinical
isolates with a MIC values close to Linezolid. In addition, analogue 9bt display a significant
activity against one clinical isolate. The rest of the active analogues presented high MIC
values (100 and 125 mg/mL), but interestingly, there are differences in the clinical isolate
target depending on their stereochemistry.
17

10bs
10aw
9bs
9aw
10bw
9bw
9bu
9bt
Figure 9. Active Linezolid dipeptide-type analogues against multidrug-resistant
Mycobacterium tuberculosis clinical isolates.
As previously described, molecular docking of 9bu shows a non-Linezolid like pose
in PTC, establishing π-π, σ-π and two hydrogen bond with G2505 (Figure 4), which is
consistent with its significant antibacterial activity. Analogue 9bs shows a similar pose than
Linezolid and establish a hydrogen bond and σ-π interaction with G2505 nucleotide, the
Fmoc group is not involve in the interaction (Figure S99). The rest of active analogues
display non-typical interactions with G2505 nucleotide and other nucleotides. For example,
analogue 9bt shows a π-π interaction between a benzyloxy ring and A2062 nucleotide, and
a hydrogen bond between the C ring-oxygen and G2583 nucleotide (Figure S100). Analogue
9bw displays two s-p interactions between Fmoc group and Guanine base in G2505
nucleotide, the rest of molecule structure is not involved. The 10bw analogue has a s-p
interaction where a terbutyl protecting group and Guanine base in G2505 nucleotide
participate (Figures S101-S102).
Table S13 lists interaction type and distances observed in the molecular docking of
Linezolid dipepetide-type analogues. As seen, the distance of donor-acceptor atoms are in
the range of 2.8 y 4.6 Å. In addition, most of Linezolid analogues interact with G2505 acting
as hydrogen bond donors, although there are some examples where act as acceptors or
establish s-p or p-p interactions. Some analogues display interaction with other nucleotides
as G2061 (10ay and its non-protected analogue), A2451 (9bu and 9bt), A2062 (9as and 9ay)
and G2583 (9bt). Molecular properties as LogPow, H-donor and H-acceptor number,
molecular weight (MW) and total polar surface area (TPSA) were calculated in order to
18

predict if these analogues might have good permeability and distribution if supplied orally
(Lipinki’s rule or the rule of 5).42,43 LogPow and TPSA values indicate these Linezolid
dipeptide-type analogues display superior lipophilicity than Linezolid, as result of an
increased of MW and the presence of protecting groups (Table S14).
3.5 Cellular viability
In order to evaluate whether Linezolid dipeptide-type analogues (10as, 10ay, 9as, 9ay,
9aw, 9ax and 9bu) induce a cytotoxic or inhibitory effect over the proliferation of ARPE-19
human cells, we carried out the cytocompatibility assays to evaluate their effect after 48 h of
treatment (Table 5).47,48 The Linezolid dipeptide-type analogues were tested until 100 µM
due to all the compounds were insoluble in the culture media at higher concentration (Figure
S103). The results showed no significant reduction on cell viability or proliferation,
remaining proliferation percentages over 80%. Interestingly, the 10ay analogue preserves the
viability until a 25 mM, which is a seven-fold the MIC concentration (3.42 mM) obtained in
the antibacterial assay. This compound present cytotoxicity at higher concentration (50 and
100µM), reducing the cellular proliferation percentage and affecting the cellular
morphology. In comparison, the cytotoxic positive control Doxorubicin, induced a strong
cytotoxic antiproliferative effect over ARPE-19 cells, reducing cell proliferation by
approximately 50% at a concentration of 0.125µM.
The exposure of the ARPE-19 cells to the Linezolid dipeptide-type analogues resulted in
the maintenance of metabolic activity, viability and cellular integrity, indicating non-
cytotoxic effects due to the Linezolid analogues treatments. Figures of micrographs depicting
the cellular morphology of ARPE-19 cells treated with the Linezolid analogues, as well as
controls are presented in supporting material (Figure S104).
Table 5. Proliferation percentages of ARPE-19 cells culture with Linezolid
dipepetide-type analogues at 50 µM.
92.9 4.5
86.3 1.5
84.3 4.6
88.8 2.9
9as
9ay
9aw
9ax
19

92.5 10.7
93.7 7.5
71.9 4.3c
85.2 4.7
9bu
10as
10ay
Linezolid
a
Data is expressed as mean of three independent experiments performed by
triplicate.
b Doxorubicin was employed as a control.
c This result was obtained at 25 mM.
4. Conclusions
Molecular docking of new Linezolid dipeptide-type analogues in PTC of E. coli used as
model predicted different interactions as s-p and p-p and different hydrogen bond
interactions including the well-known interaction of the amide hydrogen with the G2505 and
other nucleotides. The nature and number of the interactions with the active site as well as
the cooperative effect of these interactions result in high thermodynamic stability and more
favorable couplings, which may retard bacterial resistance towards these analogues. Most
active analogues display better scoring than Linezolid in the molecular docking analysis. The
results of e antimicrobial activity point analogue 10ay as the most active compound due to
its comparable or even higher activity than Linezolid against three clinical isolates of MRSA.
The tests against Mycobacterium tuberculosis strains showed 9bu analogue as potential
antituberculosis drug, due to its activity against multidrug-resistant clinical isolates with
MICs close to Linezolid. The protecting groups increase the molecular weight in an
acceptable range and their lipophilicity is highly superior to Linezolid, which may be
beneficial to the cellular penetration. Noteworthy, the analogues 9bu and 10ay preserve the
cellular integrity and metabolic viability of ARPE-19 indicating non-cytotoxic effects at
concentration highly superior to their MIC values. We are currently conducting our efforts
to the synthesis of non-protected Linezolid dipeptide-type analogues with increased
solubility in aqueous culture media. Base on the molecular docking analysis these analogues
with acceptor or donor hydrogen bond groups may increase the number of supramolecular
interactions in PTC, and thereby, result in an improve biological activity.
20

5. – Experimental section
5.1 Chemistry
Reagents and solvents obtained from commercial suppliers were used without
previous purification. The reactions with anhydrous conditions requirements were carry on
Ar atmosphere. The nuclear magnetic resonance was carried out in a Bruker Advance III HD
400 MHz equipped with an intern reference of tetramethylsilane (TMS). The FTIR spectra
were recorder on a Perkin Elmer Spectrum 400 FTIR spectrometer. The melting points were
obtained in an Electrothermal apparatus. The electrospray ionization mass spectra (ESI/MS)
were obtained on 6130 Quadrupole LC/MS of Agilent Technologies, in negative and positive
ion modes.
5.2. Synthesis of compounds
N-aryl coupling. To a mixture of the oxazolidinone (2a-b or 3a-b) (0.32 mmol), CuI (0.01
mmol), K2CO3 (0.53 mmol) and N,N´-dimethylmethanediamine (0.06 mmol) in 25 mL
toluene was added 4-(4-bromo-2-fluorphenyl)morpholine (1) (0.32 mmol). The reaction
mixture was stirred at 115°C for 72 h. Then, water (25 mL) was added to stop the reaction
and the organic phase was separated, filtered in zeolite and the solvent was evaporated. The
product purification was carried out by flash chromatography usign silica gel and a mixture
of petroleum ether and ethyl acetate (1:1) as eluent.
(R)-5-((S)-1-(dibenzylamino)ethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one
(4a). White solid, yield 75 %. M.p. 68.5-69.5 °C FTIR: 3059, 2955, 1753, 1514, 1449, 1402,
1234, 1117 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.30 (m, 10 H), 7.18 (dd, J= 11.8, 2.5 Hz,
1H), 7.00 (ddd, J= 7.1, 2.5, 1.0 Hz, 1H), 6.89 (dd, J= 9.1, 9.0 Hz, 1H), 4.38 (ddd, J= 8.3,
8.2, 7.2 Hz, 1H), 3.86 (m, 4H), 3.80 (t, J= 8.7 Hz, 1H), 3.75(d, J= 13.5 Hz, 2H), 3.57 (dd,
J= 9.0, 6.6 Hz, 1H), 3.48 (d, J= 13.5 Hz, 2H), 3.04 (m, 4H) 2.87 (dc, J= 8.9, 6.6 Hz, 1H),
13
1.28 (d, J= 6.6 Hz, 3H). C NMR (100 MHz, CDCl3): δ 155.5 (d, JC-F= 244.4 Hz), 154.6,
139.1, 136.2 (d, JC-F= 9.0 Hz) 133.3 (d, JC-F= 10.5 Hz), 128.9, 128.5, 127.4, 118.7 (d, JC-F=
4.1 Hz), 114.2 (d, JC-F= 3.3 Hz), 107.7 (d, JC-F= 25.8 Hz), 74.2, 66.9, 56.7, 54.6, 51.9, 49.4,
8.6. MS(IE) m/e 489(5), 224(100), 91(90).
21

(R)-5-((S)-1-(dibenzylamino)-2-phenylethyl)-3-(3-fluoro-4-
morpholinophenyl)oxazolidin-2-one (4b). White solid, yield 60%. FTIR: 3029, 2826,
1
1748, 1513, 1110 cm-1. H NMR (400 MHz, CDCl3): δ 7.38 (dd, J1=13.5, J2=2.6 Hz, 1H),
7.13 (m,15H), 7.13 (m, 3H), 7.04 (t, J=9.2 Hz, 1H), 4.99 (m, 1H), 4.06 (t, J=9.0 Hz, 1H),
13
3.74 (m, 4H), 3.64 (m, 6H), 3.00 (dc, 1H), 2.96 (m, 1H). C NMR (100 MHz, CDCl3): δ
155.0 (J= 245.8 Hz), 154.5, 140.3, 139.7, 127.4, 126.5, 119.7, 114.7, 107.2 (J=25.7), 72.9,
66.6, 62.7, 54.2, 51.2 (J=2.5 Hz), 49.0, 31.9. MS(IE) m/e 565(5), 474(30), 383(10), 91(80).
((S)-5-((S)-1-(dibenzylamino)ethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one
(5a). White solid, yield 70 %. M.p. 78.0-79.0 °C. FTIR: 3032, 2955, 1753, 1514, 1449, 1402,
1234, 1117 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.37 (dd, J= 11.8, 2.6 Hz, 1H), 7.30 (m, 10
H), 7.00 (ddd, J= 7.3, 2.0, 1.3 Hz, 1H), 6.94 (dd, J= 9.2, 9.0 Hz, 1H), 4.56 (ddd, J= 5.4, 5.1,
5.1 Hz, 1H), 3.93 (d, J= 13.6 Hz, 2H), 3.88 (m, 4H), 3.68 (dd, J= 8.4, 2.4 Hz, 1H), 3.48 (d,
13
J= 13.6 Hz, 2H), 3.07 (m, 4H), 2.92 (dc, J= 6.8, 5.1 Hz, 1H), 1.26 (d, J= 6.8 Hz, 3H). C
NMR (100 MHz CDCl3): δ 155.5 (d, JC-F= 244.5 Hz), 154.5, 139.6, 136.2 (d, JC-F= 8.9 Hz),
133.5 (d, JC-F= 10.5 Hz), 128.9, 128.3, 127.0, 118.7 (d, JC-F= 4.2 Hz), 113.7 (d, JC-F= 3.3 Hz),
107.3 (d, JC-F= 26.3), 76.2, 67.0, 55.1, 54.8, 51.0, 47.6, 10.2. MS(IE) m/e 489(5), 224(100),
91(90).
(S)-5-((S)-1-(dibenzylamino)-2-phenylethyl)-3-(3-fluoro-4-
morpholinophenyl)oxazolidin-2-one (5b). White solid, yield 60%. FTIR: 3029, 2826,
1
1748, 1513, 1110 cm-1. H NMR (400 MHz, CDCl3): δ 7.36 (m, 1H),7.34-7.20 (m, 15H),
7.07 (d,d, J1= 9.5 Hz, J2= 2.5 Hz, 1H), 6.95 (t, J= 9.2 Hz, 1H), 4.48 (m, 1H), 4.08 (d, 2H),
3.89 (m, 4H), 3.73 (d,d J1= 8.0 Hz, J2= 6.6 Hz, 1H), 3.55 (d, 2H), 3.47 (d,d J1= 9.1 Hz, J2=
8.3 Hz, 1H), 3.23 (m, 2H), 3.09 (m, 4H), 2.81 (d,t, J1= 10.6 Hz, J2=3.6 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 154.5, 139.3, 138.8, 136.1 (d, J= 9.0 Hz), 133.5 (d, J= 10.3 Hz), 129.5,
129.1, 128.8, 128.4, 127.2, 126.5, 118.8 (d, J= 3.6 Hz), 113.7 (d, J= 3.1 Hz), 107.2 (d, J=26.0
Hz), 72.9, 67.0, 61.2, 55.9, 51.1 (d, J= 3.6 Hz), 46.8, 30.9.
Hydrogenolysis. The compounds 4a-b or 5a-b (0.06 mmol) were dissolved in dry methanol
(50 mL) and Pd/C 10% (4.1 mmol) was added. Then, three acetic acid drops were added and
the reaction mixture was stirred for 4 h under H2 atmosphere. Then, the Pd/C was filtered
and the organic solvent was evaporated to obtain an analytical pure product.
22

(R)-5-((S)-1-aminoethyl)-3-(3-fluoro-4-morpholinphenyl)oxazolidin-2-one (6a). White
solid, yield 90 %. M.p. 196-197 ꢀC.FTIR: 3373, 2952, 2835, 1740, 1636, 1514, 1445, 1404,
1237, 1112 cm-1. 1H NMR(400 MHz, CDCl3): δ 7.45 (dd, J= 11.8, 2.6 Hz, 1H), 7.15 (ddd,
J= 8.8, 1.5, 1.2 Hz, 1H), 6.92 (dd, J= 9.1, 9.0 Hz, 1H), 4.48 (ddd, J= 8.4, 8.1, 7.2 Hz, 1H),
3.92 (dd, J= 8.5,1.6 Hz, 2H), 3.87(m, 4H), 3.34 (m, 1H), 3.05 (m, 4H) 1.19 (d, J= 6.6 Hz,
3H). 13C NMR(100 MHz, CDCl3): δ 155.5 (d, JC-F= 220.4 Hz), 154.6, 136.2 (d, JC-F= 7.3 Hz)
133.4 (d, JC-F= 10.2 Hz), 118.8 (d, JC-F= 4.1 Hz), 113.8 (d, JC-F= 3.3 Hz), 107.4 (d, 26.1),
76.2, 67.0, 51.1, 48.2, 46.2, 17.7. MS(IE) m/e 309(100), 209(90), 164(50), 44(90).
(R)-5-((S)-1-amino-2-phenylethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one
1
(6b). White solid, yield 95 %. FT-IR 3052, 2925, 1753, 1515, 1234 cm-1. H NMR (400
MHz, CDCl3): δ 7.55 (d, J1=15.1 Hz, 1H), 7.28 (m, 6H), 7.07 (t, 9.4 Hz, 1H), 4.48 (m, 1H),
3.98 (m, 2H), 3.74 (m, 4H), 3.18 (m, 1H), 2.97 (m, 4H), 2.76 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 154.6 (J= 241.1 Hz, C-F), 154.7, 138.9, 135.8, 134.2, 134.1, 129.8, 126.6, 119.7,
114.5, 107.2 (J=25.7, CH), 75.8, 66.6, 54.7, 51.2, 46.3, 35.3.
(S)-5-((S)-1-aminoethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one (7a). White
solid, yield 80 %. M.p. 128-129 ꢀC. FTIR: 3373, 2956, 1744, 1625, 1514, 1407, 1234, 1114
cm-1. 1H NMR (400 MHz, CDCl3): δ 7.51 (dd, J= 12.4, 2.6 Hz, 1H), 7.23 (dd, J= 8.8, 1.9 Hz,
1H), 7.05 (dd, J= 9.8, 9.1Hz, 1H), 4.45 (ddd, J= 8.7, 8.6, 7.8 Hz, 1H), 4.01 (dd, J= 9.0, 8.9
Hz, 1H), 3.83 (dd, J= 9.0, 8.9 Hz, 1H), 3.73 (m, 4H), 3.02 (m, 1H), 2.96 (m, 4H), 1.05 (d, J=
6.5 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 155.5 (d, JC-F= 242.1 Hz), 154.7, 135.8 (d, JC-
F= 8.8 Hz) 134.1 (d, JC-F= 10.4 Hz), 119.7 (d, JC-F= 4.2 Hz), 114.4 (d, JC-F= 3.2 Hz), 107.3
(d, JC-F= 26.0Hz), 75.8, 66.6, 51.2, 49.2, 47.1, 18.2. MS(IE) m/e 309(100), 209(90), 164(50),
44(90).
(S)-5-((S)-1-amino-2-phenylethyl)-3-(3-fluoro-4-morpholinophenyl)oxazolidin-2-one
(7b). White solid, yield 95%. FT-IR 3050, 2923, 1751, 1513, 1232 cm-1. 1H NMR (400 MHz,
CDCl3): δ 7.55 (d, J1=15.1 Hz, 1H), 7.28 (m, 6H), 7.07 (t, 9.4 Hz, 1H), 4.48 (m, 1H), 3.98
13
(m, 2H), 3.74 (m, 4H), 3.18 (m, 1H), 2.97 (m, 4H), 2.76 (m, 2H). C NMR (100 MHz,
CDCl3): δ 154.6 (J= 241.1 Hz), 154.7, 138.9, 135.8, 134.2, 134.1, 129.8, 126.6, 119.7, 114.5,
107.2 (J=25.7), 75.8, 66.6, 54.7, 51.2, 46.3, 35.3.
23

Peptide coupling with protected L-amino acids. N-Fmoc α-amino acids (8s-z) (0.13 mmol)
were dissolved in DMF (10 mL) and then DIC (0.15 mmol) and HOBt (0.15 mmol) were
added. The mixture was stirred for 20 min and then the compound (6a-b or 7a-b) (0.13 mmol)
was added. The reaction mixture was stirred for 12 h. Distilled water was added to stop the
reaction and a solid product was filtered and repeatedly washed with water. The compounds
were dried under vacuum at RT.
(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((R)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (9as). White
solid, yield 55 %. M.p. 135.5-136.5 ꢀC FTIR: 3293 3023, 2955, 1754, 1699, 1656, 1514,
1448, 1226, 1106 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J= 7.5 Hz, 2H), 7.52 (t, J=
6.5 Hz, 2H), 7.37 (m, 3H), 7.28 (m, 5 H), 7.18 (m, 2H), 7.06 (m, 1H), 6.89 (t, J= 9.1, 1H),
6.35 (sa, 1H), 5.40 (sa, 1H), 4.43 (m, 3H), 4.29 (m, 1H), 4.17 (t, J= 6.7 Hz, 1H), 4.13 (m,
1H), 3.85 (m, 4H), 3.75 (t, J= 9.1 Hz, 1H), 3.56 (m, 1H), 3.04 (m, 6H),1.15 (d, J= 6.3 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 170.1, 156.0, 155.9, 155.5 (d, JC-F= 246.1 Hz), 154.6,
143.6, 141.3, 136.7 (d, JC-F= 8.6 Hz), 136.1, 132.8 (d, JC-F= 10.1 Hz), 129.2, 128.7, 127.7,
127.2, 127.0, 125.0, 120.0, 118.8 (d, JC-F= 4.1 Hz), 114.0 (d, JC-F= 3.2 Hz), 107.5 (d, 26.1),
74.3, 67.1, 66.9, 56.3, 51.0, 47.8, 47.7, 47.1, 38.0, 14.4. MS(ESI): 679 [M+H]+, 701
[M+Na]+, 717 [M+K]+.
(9H-fluoren-9-yl)methyl
((S)-3-(4-(tert-butoxy)phenyl)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)ethyl)amino)-1-oxopropan-2-yl)carbamate
(9at). White solid, yield 38 %. M.p. 115.5-116.5 ꢀC FTIR: 3294, 3023, 2976, 1748, 1710,
1655, 1513, 1448, 1227, 1105 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.72 (d, J= 7.5 Hz, 2H),
7.53 (dd, J= 7.2, 6.9 Hz, 2H), 7.40 (dd, J= 12.1, 2.1 Hz, 1H), 7.36 (m, 2 H), 7.26 (m, 2H),
7.08 (d, J= 6.8, 2H), 7.01 (d, J= 8.3 Hz, 1H), 6.89 (d, J= 7.6 Hz, 3H), 5.87 (sa, 1H), 4.48 (m,
1H), 4.32 (m, 3H), 4.13 (m, 2H), 3.83 (m, 4H), 3.76 (t, J= 8.7 Hz, 1H), 3.57 (t, J= 6.4 Hz,
13
1H), 3.00 (m, 6H), 1.26 (sa, 9H),1.13 (d, J= 5.8 Hz, 3H). C NMR (100 MHz, CDCl3): δ
171.0, 156.0, 155.5 (d, JC-F= 245.1 Hz), 154.5, 154.4 143.6, 141.2, 136.4 (d, JC-F= 8.9 Hz),
133.0 (d, JC-F= 10.4 Hz), 131.0, 129.2, 128.7, 127.8, 127.5, 127.0, 124.9, 124.2 119.9, 118.8
(d, JC-F= 4.1 Hz), 114.0 (d, JC-F= 3.2 Hz), 107.5 (d, JC-F= 26.1), 78.4, 74.3, 67.1, 66.9, 56.3,
51.0, 47.8, 47.7, 47.1, 38.4, 28.7, 14.4. MS(ESI): 751 [M+H]+, 773 [M+Na]+, 789 [M+K]+.
24

(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((R)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)ethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (9au). White
solid, yield 38 %. M.p. 102.0-103.0 ꢀC.FTIR: 3295, 3023, 2956, 1749, 1716, 1656, 1514,
1448, 1226, 1106 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J= 7.4 Hz, 2H), 7.71 (m,
1H), 7.58 (d, J= 7.2 Hz, 2H), 7.41 (t, J= 7.5 Hz, 2H), 7.37 (m, 1H), 7.32 (t, J= 7.5 Hz, 2 H),
7.06 (m, 1H), 6.41 (sa, 1H), 5.21 (sa, 1H), 4.60 (m, 1H), 4.37 (m, 2H), 4.13 (m, 3H), 4.12
(m, 4H), 4.00 (m, 1H), 3.78 (m, 1H), 3.38 (m, 4H), 1.65 (m, 1H), 1.54 (m, 2H), 1.25 (d, J=
5.9 Hz, 3H), 0.95 (sa, 6H). 13C NMR (100 MHz, CDCl3): δ 172.3, 155.5 (d, JC-F= 254.2 Hz),
155.0, 154.5, 143.6, 141.2, 136.4 (d, JC-F= 7.0 Hz), 133.0 (d, JC-F= 10.0 Hz), 127.8, 127.0,
124.8, 120.0, 118.8 (d, JC-F= 5.4 Hz), 114.0 (d, JC-F= 4.0 Hz), 107.8 (d, JC-F= 25.1 Hz), 74.7,
67.0, 65.4, 53.6, 52.3, 47.7, 47.5, 47.1, 41.1, 24.7, 22.9, 14.3. MS(ESI): 645 [M+H]+, 667
[M+Na]+, 683 [M+K]+.
(9H-fluoren-9-yl)methyl
((2S)-3-(tert-butoxy)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)ethyl)amino)-1-oxobutan-2-yl)carbamate
(9aw). White solid, yield 35 %. M.p. 84.5-86.5.0 ꢀC FTIR: 3300, 3023, 2973, 1749, 1719,
1
1670, 1514, 1448, 1224, 1048 cm-1. H NMR (400 MHz, CDCl3): δ 7.77 (dd, J= 7.4, 5.4 Hz,
2H), 7.61 (d, J= 7.5 Hz, 3H), 7.42 (dd, J= 7.3, 7.0 Hz, 3H), 7.33 (dd, J= 7.5, 7.0 Hz, 2H),
7.10 (m, 1H), 5.09 (sa, 1 H), 4.64 (m,1H), 4.41 (m, 2H), 4.25 (m, 2H), 4.18 (m, 2H), 4.01
(m, 5H), 3.86 (m,1H), 3.24 (m, 4H), 1.29 (s, 9H), 1.24 (s, 3H), 1.08 (d, J= 6.3 Hz, 3H). 13C
NMR (100 MHz, CDCl3): δ 169.7, 156.0, 155.9, 155.4 (d, JC-F= 258.1 Hz), 143.6, 141.3,
127.8, 127.0, 125.0, 120.0, 119.9 (d, JC-F= 7.4 Hz), 113.8 (d, JC-F= 3.4 Hz), 107.7 (d, 26.4),
75.7, 74.5, 66.9, 66.5, 66.1, 58.9, 51.7, 47.8, 47.7, 47.2, 28.2, 17.2, 14.7. MS(ESI): 689
[M+H]+, 711 [M+Na]+, 729 [M+K]+.
(9H-fluoren-9-yl)methyl
((R)-3-(tert-butylthio)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)ethyl)amino)-1-oxopropan-2-yl)carbamate
(9ax). White solid, yield 36 %. M.p. 91.5-92.5 ꢀC. FTIR: 3296, 3008, 2951, 1754, 1714,
1675, 1514, 1232, 1110 cm-1. 1H NMR (CDCl3, 400 MH z): δ 7.74 (m, 2H), 7.55 (m, 2H), 7.
39 (m, 9H), 7.26 (m, 8H), 7.20 (m, 3H), 7.01 (d, J= 9.2 Hz, 1H), 6.83 (t, J= 9.1 Hz, 1H), 6.00
(d, J= 8.6 Hz, 1H), 5.00 (d, J= 7.3 Hz, 1H), 4.41 (m, 1H), 4.20 (m, 3H), 4.12 (m, 1H), 3.91(t,
J= 9,1 Hz, 1H), 3.80 (m, 4H), 3.75 (dd, J= 7.0, 6.9 Hz, 1H), 3.69 (dd, J= 9.0, 6.6 Hz, 1H),
25

3.02 (m, 4H), 2.62 (d, J= 6.5 Hz, 2H), 1.18 (d, J= 6.7 Hz, 3H). 13C NMR (CDCl3, 100 MHz):
δ 170.0, 160.0 (d, J= 233.2 Hz), 155.9, 155.7, 144.0, 143.5, 141.3, 136.0 (d, J= 13.0 Hz),
133.6 (d, J= 12.1 Hz), 129.4, 128.1, 127.8, 127.1, 127.0, 124.9, 120.0, 118.3 (d, J= 6.1 Hz),
113.0 (d, J= 3.1 Hz), 107.7 (d, J= 29.3 Hz), 79.2, 74.5, 68.1, 66.9, 54.8, 51.0, 47.9, 47.7,
47.0, 30.4, 14.0.
(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((R)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)ethyl)amino)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-
yl)carbamate (9ay). White solid, yield 36 %. M.p. 108.5-109.5 ꢀC. FTIR: 3304, 3059, 2955,
1750, 1718, 1671, 1513, 1228, 1114 cm-1. 1H NMR (CDCl3, 400 MH z): δ 7.70 (m, 3H), 7.50
(m, 3H), 7. 36 (m, 3H), 7.30 (m, 6H), 7.24 (m, 2H), 7.21 (m, 9H), 6.94 (m, 1H), 6.70 (m,
1H), 6.43 (sa, 1H), 5.07 (sa, 1H), 4.50 (m, 1H), 4.31 (m, 1H), 4.21 (m, 2H), 4.09 (m, 1H),
3.80 (m, 5H), 3.75 (m, 2H), 2.93 (m, 4H), 2.57 (m, 2H), 1.27 (d, J= 5.6 Hz, 3H). 13C NMR
(CDCl3, 100 MHz): δ 170.6, 155.7, 155.5 (d, J= 244.2 Hz), 154.1, 143.6, 141.2, 138.7, 136.2
(d, J= 8.8 Hz), 133.1 (d, J= 9.6 Hz), 132.4, 130.9, 129.4, 128.7, 128.1, 127.7, 127.0, 126.9,
125.0, 119.9, 118.7 (d, J= 4.3 Hz), 113.9 (d, J= 2.0 Hz), 107.5 (d, J= 27.5 Hz), 85.9, 74.8,
67.4, 66.9, 54.3, 50.9, 47.4, 47.0, 45.7, 33.6, 17.7. MS(ESI): [M+H]+= 912 amu, [M+Na]+=
934 amu, [M+K]+= 950 amu.
(9H-fluoren-9-yl)methyl
((S)-3-(tert-butoxy)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)ethyl)amino)-1-oxopropan-2-yl)carbamate
(9az). White solid, yield 36 %. M.p. 80.0-81.0 ꢀC. FTIR: 3296, 3020, 2969, 1736, 1715,
1669, 1514, 1225, 1114 cm-1. 1H NMR (CDCl3, 400 MH z): δ 7.73 (d, J= 7.5 Hz, 2H), 7.52
(d, J= 9.4 Hz, 2H), 7. 38 (m, 2H), 7.30 (m, 3H), 7.00 (m, 1H), 6.84 (m, 1H), 4.65 (m, 1H),
4.22 (m, 3H), 4.15 (m, 2H), 3.82 (m, 6H), 3.75 (dd, J= 9.2, 8.9 Hz, 1H), 3.37 (dd, J= 8.9, 8.6
Hz, 1H), 2.97 (m, 4H), 1.37 (d, J= 6.9 Hz, 3H). 13C NMR (CDCl3, 100 MHz): δ 171.0, 156.5
(d, J= 225.2 Hz), 156.1, 154.6, 143.9, 141.2, 137.0 (d, J= 7.3 Hz), 133.5 (d, J= 7.1 Hz), 127.6,
127.0, 125.0, 119.9, 118.8 (d, J= 4.5 Hz), 114.1 (d, J= 4.0 Hz), 107.6 (d, J= 20.0 Hz), 78.2,
75.2, 68.1, 66.8, 61.5, 56.1, 50.9, 47.7, 47.1, 46.3, 27.3, 17.9. MS(ESI): [M+H]+= 675 amu,
[M+Na]+= 697 amu, [M+K]+= 713 amu.
(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((R)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate
26

(9bs). White solid, yield 48%. M.p. 87-88 ꢀC. FTIR: 3309, 3035, 2960, 1775, 1760, 1684,
1517, 1448, 1221 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.74(d, J= 7.6 Hz, 2H), 7.51 (d, J=9.6
Hz, 2H), 7.40-7.13 8m, 14H), 7.39 (d, J=7.9 Hz, 2H), 7.35 (m, 1H), 7.29 (m, 1H), 7.05 (d,
J= 7.6 Hz, 1H), 6.91(t, J= 9.2 Hz, 1H), 4.61 (m, 1H), 4.39 (m, 1H), 4.32 (m, 1H), 4.24 (m,
2H), 4.20 (m,1H), 3.94(dd, J= 8.7, J=8.6 Hz, 1H), 3.86 (m, 4H), 3.66(dd, J= 8.3, J=8.2 Hz,
1H), 3.05 (m, 4H), 3.12 (m, 1H), 3.00 (m, 1H), 2.94 (m, 1H), 2.88 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 170.9, 155.0 (d, JC-F= 233 Hz), 154.9, 154.1, 143.7 (2 x C), 141.3 (2 x C),
136.9, 136.1, 136.0, 132.1, 130.9, 129.3 (2 x CH), 129.4 (2 x CH), 129.2 (2 x CH), 128.9 (2
x CH), 128.7, 127.8 (2 x CH), 127.1 (2 x CH), 125.1 (2 x CH), 120.0 (2 x CH), 118.8 (d, JC-
F=3.8 Hz), 113.9 (d, JC-F= 3.2 Hz), 107.4 (d, JC-F=26.3 Hz), 73.1, 68.2, 66.9 (2 x CH2), 52.5,
52.2, 51.1 (2 x CH2), 48.8, 48.0, 38.8, 37.0. MS(ESI): 756 [M+2H]+.
(9H-fluoren-9-yl)methyl
((S)-3-(4-(tert-butoxy)phenyl)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)-2-phenylethyl)amino)-1-oxopropan-2-
yl)carbamate (9bt). White solid, yield 43%. M.p. 77-79 ꢀC. FTIR: 3310, 3067, 2978, 1752,
1717, 1670, 1515, 1224, 1046 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.83 (d, J=7.5 Hz, 2H),
7.68 (t, J= 7.6Hz, 2H), 7.53 (m, 2H), 7.31 (m, 2H), 7.18 (m, 5H), 6.92 (m, 1H), 4.48 (m, 1H),
4.30 (m, 1H), 4.29 (m, 1H), 4.14 (m, 1H), 3.77 (m, 1H), 3.73 (m, 1H), 3.54 (m, 1H), 3.15 (m,
1H), 1.15 (sa, 1H). 13C NMR (100 MHz, CDCl3): δ 176.7, 156.6, 155.6(d, JC-F=244.0), 154.7,
154.3, 143.8, 141.2, 139.0, 136.5, 135.4(d, JC-F=9.0), 132.5(d, JC-F=10.3), 129.4, 129.2,
128.8, 127.6, 127.1, 127.0, 125.0, 124.3, 119.9, 118.7(d, JC-F=2.9), 114.1(d, JC-F=3.7),
107.7(d, JC-F=26.0), 78.4, 72.4, 67.3, 64.5, 56.6, 53.1, 51.4, 47.3, 46.9, 38.0, 37.1, 28.8.
MS(ESI): 826 [M+H]+.
(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((R)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)-2-phenylethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate
(9bu). White solid, yield 49%. M.p. 120-122 ꢀC. FTIR: 3305, 3064, 2959, 1757, 1715, 1686,
1516, 1225, 1031 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J=7 Hz, 2H), 7.42 (m, 1H),
7.40 (m, 2H), 7.33 (m, 2H), 7.32 (m, 2H), 7.22-7.13 (m, 5H), 7.01 (d, J=7.1 Hz, 1H), 6.85 (t,
J=9.1Hz, 1H), 4.56 (m,1H), 4.50 (m, 2H), 4.43 (m, 1H), 4.21 (m, 1H), 4.04 (m, 1H), 3.90
(m, 1H), 3.88 (m, 4H), 3.81 (m, 1H), 3.10 (dd, J=14.3, 4.4 Hz, 1H), 3.05 (m, 4H), 2.90 (dd,
13
J=14.0, 9.8, 1H), 1.54 (m, 2H), 1.34 (m, 1H), 0.91-0.79 (m, 6H). C NMR (100 MHz,
27

CDCl3): δ 172.4, 155.5 (d, JC-F= 246 Hz), 154.3 (C=O), 154.2, 143.7, 141.4, 136.6, 136.0,
132.7, 129.2 (2 x CH), 128.7(2 x CH), 127.9, 127.7, 127.0, 124.9, 120.1, 118.8 (d, JC-F =4
Hz), 114.1 (d, JC-F =3 Hz), 107.7 (d, JC-F =26 Hz), 73.4, 67.0, 66.9 (2 x CH2), 53.4, 52.8,
51.0 (d, JC-F=3 Hz, 2 x CH2), 48.0, 47.2, 40.5, 38.8, 24.7, 22.8, 21.8. MS(ESI): 739
[M+H2O]+.
(9H-fluoren-9-yl)methyl
((2S)-3-(3a,7a-dihydro-1H-indol-3-yl)-1-(((S)-1-((R)-3-(3-
fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)-2-phenylethyl)amino)-1-
oxopropan-2-yl)carbamate (9bv). White solid, yield 40%. M.p. 123-125 ꢀC. FTIR: 3298,
3063, 2961, 1758, 1726, 1682, 1515, 1229, 1031 cm-1. 1H NMR(400 MHz, CDCl3): δ 7.74
(d, J=7.6 Hz, 1H), 7.67 (m, 2H), 7.42 (m, 2H), 7.31 (m, 2H), 7.21 (m, 2H), 7.16 (m, 1H),
7.03 (m, 8H), 6.69 (m, 1H), 4.41 (m, 1H), 4.31 (m, 2H), 4.20 (m, 1H), 4.14 (m, 1H), 4.05 (m,
1H), 3.59 (m, 1H),3.46 (m, 1H), 3.07 (m, 1H), 2.90 (m, 1H). 13C NMR (100 MHz, CDCl3):
δ172.8, 155.0(d, JC-F=249.5), 154.7, 154.6, 144.0, 143.9, 143.6, 141.3, 136.6(d, JC-F=9.0),
136.5, 136.3, 132.5(d, JC-F=12.0), 130.9, 129.2, 128.8, 128.7, 127.6, 127.1, 127.0, 125.1,
122.7, 119.9, 118.7(d, JC-F=6.0), 114.1(d, JC-F=2.0), 107.6(d, JC-F=26.9), 72.4, 67.0, 66.9,
55.8, 51.0, 50.9, 47.3, 47.0, 42.3, 38.8. MS(ESI): 796 [M+H]+.
(9H-fluoren-9-yl)methyl
((2S)-4-(tert-butoxy)-1-(((S)-1-((R)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)-2-phenylethyl)amino)-1-oxopentan-2-
yl)carbamate (9bw). White solid, yield 43%. M.p. 89-91 ꢀC. FTIR: 3304, 3063, 2972, 1756,
1721, 1680, 1514, 1224, 1032 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J=7.4 Hz, 2H),
7.50 (t, J=7.5, 2H), 7.39 (t, J=7.3 Hz, 2H), 7.31 (m, 2H), 7.22-7.07 (m, 5H), 7.01 (m, 1H),
6.88 (m, 1H), 6.71 (m,2H), 5.39 (brs, 1H), 5.34 (brs, 1H), 4.43 (m, 1H), 4.33 (m, 1H), 4.15
(m, 2H), 3.88 (m, 1H), 3.86 (m, 4H), 3.50 (m, 2H), 3.03 (m, 4H), 2.81 (m, 2H), 1.29 (s,
9H),1.25 (s, 3H).13C NMR (100 MHz, CDCl3): δ 171.5, 154.2, 153.4, 143.7, 141.4, 136.2,
135.6, 129.3, 128.7, 127.8, 127.8, 127.1, 126.9, 124.9, 120.1, 118.6, 114.2 (d, JC-F =3 Hz),
107.8 (d, JC-F= 26 Hz), 72.7, 68.2, 67.1, 66.9, 55.5, 52.2, 51.0, 47.7, 47.1, 37.0, 28.2, 14.7.
MS(ESI): 765 [M+H]+.
(9H-fluoren-9-yl)methyl
((S)-1-(((S)-1-((S)-3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (10as). White
solid, yield 40%. M.p. 105.5-106.5 ꢀC. 1H NMR (400 MHz, CDCl3): δ 7.70 (d, J= 7.5 Hz,
28

2H), 7.41 (t, J= 7.5 Hz, 2H), 7.38 (m, 3H), 7.22 (m, 2H), 7.10 (m, 6H), 6.89 (m, 1H), 5.63
(d, J= 7.5 Hz, 1H), 4.57 (m, 1H), 4.50 (m, 1H), 4.37 (m, 1H), 4.25 (m, 1H), 4.15 (m, 1H),
4.06 (m, 1H), 3.79 (m, 3H), 3.56 (m, 1H), 2.92 (m, 6H), 1.65 (m, 1H), 1.26 (d, J= 6.9 Hz,
3H). 13C NMR (100 MHz, CDCl3): δ 172.3, 155.9, 155.5 (d, JC-F= 244.1 Hz), 154.9, 143.7,
141.2, 136.4 (d, JC-F= 8.9 Hz), 132.9 (d, JC-F= 9.2 Hz), 127.6, 127.0, 125.0, 120.0, 118.8 (d,
JC-F= 3.7 Hz), 114.0 (d, JC-F= 3.2 Hz), 107.5 (d, JC-F= 26.3 Hz), 75.2, 67.2, 66.9, 56.2, 50.9,
47.4, 47.1, 46.3, 38.0, 17.2. MS(ESI): 679 [M+H]+, 701 [M+Na]+, 717 [M+K]+.
(9H-fluoren-9-yl)methyl
((S)-3-(4-(tert-butoxy)phenyl)-1-(((S)-1-((S)-3-(3-fluoro-4-
morpholinophenyl)-2-oxooxazolidin-5-yl)ethyl)amino)-1-oxopropan-2-yl)carbamate
(10at). White solid, yield 40%. M.p. 98.5-99.5 ꢀC 1H NMR (400 MHz, CDCl3): δ 7.72 (m,
2H), 7.44 (m, 2H), 7.43 (m, 1H), 7.37 (m, 2H), 7.27 (m, 2H), 7.03 (m, 2H), 6.87 (m, 3H),
6.20 (d, J= 8.6 Hz, 1H), 4.63 (m, 1H), 4.38 (m, 3H), 4.18 (m, 1H), 4.09 (m, 1H), 3.81 (m,
3H), 3.69 (m, 1H), 2.93 (m, 6H), 1.38 (m, 9H), 1.27 (d, J= 6.3 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 176.8, 155.9, 155.5 (d, JC-F= 254.2 Hz), 154.7, 143.5, 141.2, 136.7 (d, JC-F= 8.2
Hz), 131.7 (d, JC-F= 9.3 Hz), 127.6, 127.0, 125.0, 119.9, 118.7 (d, JC-F= 4.4 Hz), 114.0 (d, JC-
F= 3.7 Hz), 107.8 (d, JC-F= 30.7 Hz), 78.5, 74.7, 67.3, 66.9, 54.9, 50.8, 47.3, 47.2, 46.9, 37.2,
28.8, 17.4. MS(ESI): 751 [M+H]+, 773 [M+Na]+, 789 [M+K]+.
(9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxa-
zolidin-5-yl)ethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (10au). White solid,
yield 38 %. M.p. 110.5-111.5 ꢀC 1H NMR (400 MHz, CDCl3): δ 7.70 (m, 2H), 7.49 (m, 3H),
7.34 (m, 2H), 7.22 (m, 2H), 7.01 (m, 1H), 6.81 (m, 1H), 6.44 (sa, 1H), 4.63 (m, 1H), 4.33
(m, 3H), 4.20 (m, 2H), 3.93 (t, J= 8.9 Hz, 1H), 3.82 (m, 4H), 3.78 (m, 1H), 2.96 (m, 4H),
1.73 (m, 2H), 1.59 (m, 1H), 1.31 (d, J= 5.8 Hz, 3H), 0.69 (d, J= 6.2 Hz, 3H), 0.66 (d, J= 6.3
13
Hz, 3H). C NMR (100 MHz, CDCl3): δ 171.0, 156.1, 155.5 (d, JC-F= 245.2 Hz), 154.5,
143.6, 141.2, 136.4 (d, JC-F= 7.0 Hz), 133.0 (d, JC-F= 10.0 Hz), 154.7, 1143.8, 141.2, 127.6,
127.0, 125.0, 119.9, 118.8, 114.0, 107.5 (d, JC-F= 26.4 Hz), 75.9, 67.0, 66.9, 53.6, 50.9, 47.5,
47.0, 46.8, 42.0, 24.6, 22.4, 21.4, 14.5. MS(ESI): 645 [M+H]+, 667 [M+Na]+, 683 [M+K]+.
(9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((S)-3-(3-fluoro-4-morpholinophenyl)-2-oxooxa-
zolidin-5-yl)ethyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (10av). White
solid, yield 36 %. M.p. 106.5-107.5 ꢀC 1H NMR (400 MHz, CDCl3): δ 7.75 (t, J= 9.1 Hz,
29